BMC Medical Genetics BioMed Central Open Access Research article Deletions in the Y-derived amelogenin gene fragment in the Indian population VK Kashyap*1,2, Sanghamitra Sahoo1, T Sitalaximi1 and R Trivedi1 Address: 1National DNA Analysis Centre.Central Forensic Science Laboratory, Kolkata, INDIA and 2National Institute of Biologicals, A-32, Sector 62, Institutional Area, Noida 201307, Uttar Pradesh, India Email: VK Kashyap* - vkk2k@hotmail.com; Sanghamitra Sahoo - sanghamitra_sahoo2001@yahoo.com; T Sitalaximi - ssita2k@yahoo.com; R Trivedi - trivedi_r@hotmail.com * Corresponding author Published: 10 April 2006 BMC Medical Genetics2006, 7:37 doi:10.1186/1471-2350-7-37 Received: 21 November 2005 Accepted: 10 April 2006 This article is available from: http://www.biomedcentral.com/1471-2350/7/37 © 2006Kashyap et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Rare failures in amelogenin-based gender typing of individuals have been observed globally. In this study, we report the deletion of a large fragment of the amelogenin gene in 10 individuals out of 4,257 male samples analyzed from 104 different endogamous populations of India. Methods: Samples were analyzed using commercial genetic profiling kits. Those that exhibited failures in amelogenin-based gender identification were further analyzed with published as well as newly designed primers to ascertain the nature and extent of mutation. Results: The failure rate among Indian males was 0.23 %. Though the exact size and nature of the deletion (single point mutations at a number of positions or a single large deletion) could not be determined in the present study, it is inferred that the deletion spans a region downstream of the reverse primer-binding site of commercially available amelogenin primer sets. Deletions were conspicuously absent among the Mongoloid tribes of Northeast India, while both caste and tribal groups harbored these mutations, which was predominantly among the Y-chromosomes belonging to J2 lineage. Conclusion: Our study indicates that the different amelogenin primer sets currently included in genetic profiling multiplex kits may result in erroneous interpretations due to mutations undetectable during routine testing. Further there are indications that these mutations could possibly be lineage-specific, inherited deletions. Background Genotyping the X-Y homologous amelogenin gene segment for gender identification is widely used for DNA profiling in DNA databasing, forensic casework, archeological specimens, preimplantation and prenatal diagnoses [1-4]. The amelogenin gene is a single copy gene, homologues of which are located on Xp22.1-Xp22.3 and Yp 11.2 [5]. Regions on this gene that are sufficiently conserved are amplified for simultaneous detection of the X and Y alleles in gender identification procedures. Primers bind to the first intron region of the amelogenin gene on the X and Y-chromosomes [6] and amplify regions that differ in base sequence, hence resulting in products that are easily distinguishable by differences in size and sequence. The most widely used primer set [6] delimits a 6 bp deletion on the X-chromosome and produces fragments of 106 bp and 112 bp for the X and Y chromosomes respectively. Presence of two amplified products indicates Page 1 of 7 (page number not for citation purposes) BMC Medical Genetics 2006, 7:37 http://www.biomedcentral.com/1471-2350/7/37 provide evidence for a plausible inherited mode of transmission of the mutation. Methods • Khatri • Agharia • Maratha • Desasth Brahmin • Khandayat • Paroja • Kuruva • Iyengar Brahmin • Kallar Population samples analyzed DNA was isolated by standard organic extraction method [12] either from blood or buccal swabs of consenting 4,257 male and 2,957 female individuals belonging to 104 different endogamous groups. Individuals represented major caste and tribal groups of India, which were sampled from across 20 geographical regions of India (Table 1, Figure 1). • Vanniyar Figure Map of samples depicting 1 where the amelogenin regions covered deletion in the were study observed and location Map depicting the regions covered in the study and location of samples where amelogenin deletion were observed. a male genotype, while a single amplicon implies female genotype. However, mutations in the Y-derived fragment of the gene may result in amplification failure of the Yallele, causing misidentification of the biological sample as of a female. Similarly, mutations on the X homologue would also result in non-amplification of the X-derived fragment although the genotype would still be identified as male due to amplification of the Y-amelogenin allele. Recently, a few studies have revealed misidentification of the male genotype while employing the amelogenin gender test [[7-9]]. Failures in accurate determination of gender have been reported to be particularly high among individuals of Indian origin. The frequency of failure was observed to be 8 % by Santos et al. [7], while Thangaraj et al. [9] reported 5 cases of amelogenin failure (1.85 %) among the 270 Indian males studied. The failure rate of the amelogenin sex test was particularly high (3.6 %) in an Indian population group from Malaysia [10]. However, a parallel study testing a larger number of individuals from the Austrian National DNA database reported a failure rate of 0.018 % [11]. The high frequency in incidence of failures in the Indian sub-continent prompted us to scrutinize the amelogenin typing results of 7,214 individuals (including 4,257 males) belonging to 104 different endogamous populations that were genotyped as part of our DNA databasing project. Individuals were sampled from diverse geographic regions across India such that all existing socio-ethnic groups and linguistic families were represented. In this paper, we report failures in genotyping of male individuals due to mutations originating in the Y-homologue of the amelogenin gene. We have further characterized the nature and extent of mutations and Amelogenin typing using commercial genotyping kits The DNA samples were amplified using commercial multiplex short tandem repeat (STR) kits; PowerPlex® 16 system (Promega Corporation, Madison, USA) and Identifiler™ (Applied Biosystems, Foster City, CA), which include the amelogenin marker for gender determination. Genotyping of the amplified products was performed on an ABI Prism™ 377 DNA Sequencer (PE Applied Biosystems, Foster City, CA). The amelogenin profile was determined from the electropherograms by comparing the presence or absence of 106 and 112 bp peaks with known male and female controls. Females exhibit a single peak of 106 bp while males exhibit two peaks of 106 and 112 bps. Amelogenin typing using newly designed primers and other published primers Samples that showed abnormal amelogenin peak profiles with the commercial kits were reamplified with primers described by Steinlechner et al. [11] followed by genotyping as described above. Male samples exhibit two peaks of 219 and 225 bp, while female samples exhibit a single peak at 219 bp. Additional primers were designed for amplifying the region identified by Roffey et al. [8] to decipher the origin of mutations. The sequences of new primers designed to facilitate detection of mutation are as follows: 1. #P1: 5'- TTACGGCCATATTTAGGA-3' (for amplification of X and Y homologues) 2. #P2: 5'- GAAAGAGTCAATCCGAATGGT-3' (for amplification of Y homologue) Analysis of SRY, Y-STRs and Y-SNPs To confirm the gender of the studied samples, a sex-determining locus (SRY) [13] specific to males, was amplified. Occurrence of a single 93 bp amplicon would distinguish an authentic male DNA sample from a female DNA sample. Further, four Y- short tandem repeats (Y-STRs) [14,15] (DYS19, DYS389I, DYS389II and DYS390) were amplified to determine the extent of deletion of the Y- Page 2 of 7 (page number not for citation purposes) BMC Medical Genetics 2006, 7:37 http://www.biomedcentral.com/1471-2350/7/37 Table 1: Incidences in failures of gender testing among the 104 endogamous populations of India tested for amelogerin gene efficacy S.No State Population Social Group Individuals Tested Failure in Gender Analysis (No of samples) 1 Jammu & kashmir 2 3 Himachal Pradesh Uttaranchal 4 Uttar Pradesh 5 Bihar 6 7 Gujarat Maharastra 8 Chattisgarh 9 Jharkhand 10 Westbengal 11 Orrisa 12 Andhra Pradesh Balti Drokpa Argon Buddhist HPRajput Tharu Jaunsari Bhoksha Kanyakubj Brahmin UP Jat UP Thakur Khatri UP Kurmi Bihar Brahmin Bhumihar Rajput Kayasth Yadav Kurmi Baniya Gujarat Patel Desasth Brahmin Chitpavan Brahmin Maratha Dhangar Pawara Katkari Madia Gond Mahadeo Koli Brahmin Satnami Teli Dheria Gond Agharia Oroan Ho Bhumij Kharia Munda Birhor Santhal Oroan Brahmin Kayasth Mahishya Namasudra Bauri Maheli Karmali Kora Lodha Oriya Brahmin Karan Khandayat Gope Paroja Juang Saora Andhra Brahmin Tribe Tribe Tribe Religious Group Caste Tribe Tribe Tribe Caste Caste Caste Caste Caste Caste Caste Caste Caste Caste Caste Caste Caste Caste Caste Caste Caste Tribe Tribe Tribe Tribe Caste Caste Caste Tribe Tribe Tribe Tribe Tribe Tribe Tribe Tribe Tribe Tribe Caste Caste Caste Caste Caste Tribe Tribe Tribe Tribe Caste Caste Caste Caste Tribe Tribe Tribe Caste 67 38 51 156 50 45 45 42 98 48 48 47 45 59 65 58 53 44 50 45 45 70 78 65 150 82 95 45 45 51 50 50 35 70 42 50 56 83 64 61 61 60 110 103 60 55 54 49 51 59 99 57 62 62 60 78 50 35 106 Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil 1 Nil Nil Nil Nil Nil Nil Nil Nil Nil 1 Nil 1 Nil Nil Nil Nil Nil Nil Nil Nil Nil 1 Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil 1 Nil 1 Nil Nil Nil Page 3 of 7 (page number not for citation purposes) BMC Medical Genetics 2006, 7:37 http://www.biomedcentral.com/1471-2350/7/37 Table 1: Incidences in failures of gender testing among the 104 endogamous populations of India tested for amelogerin gene efficacy 13 Tamil Nadu 14 15 Kerala Karnataka 16 Sikkim 17 Mizoram 18 19 Arunachal Pradesh Manipur 20 Andaman & Nicobar Islands Raju Kappu Naidu Kamma Chaudhary Reddy Komati Yerukula Chenchu Naikpod Gond Lambadi Golla Sakunupakshollu Chakkiliar Tanjore Kallar Vanniyar Pallar Gounder Irular Paraiyar Nair Iyenger Brahmin Lingayat Gowda Bhovi Christian Muslim Kuruva Bhutia Nepali Lepcha Mara Hmar Lai Lusei Kuki Adi Pasi Garo Meitei Naga Hmar Manipuri Muslim Great Andamanese Caste Caste Caste Caste Caste Tribe Tribe Tribe Tribe Caste Caste Caste Caste Caste Caste Caste Tribe Caste Caste Caste Caste Caste Caste Religious Group Religious Group Tribe Tribe Caste Tribe Tribe Tribe Tribe Tribe Tribe Tribe Tribe Tribe Tribe Tribe Religious Group Tribe 66 104 106 107 104 101 100 104 107 65 30 49 101 87 33 56 54 21 87 65 98 56 52 55 65 60 75 110 48 90 80 92 92 105 203 110 105 106 101 101 24 Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil 1 1 Nil Nil Nil Nil Nil 1 Nil Nil Nil Nil Nil 1 Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Jarawa Onge Nicobarese Shompen Tribe Tribe Tribe Tribe 50 16 28 33 Nil Nil Nil Nil * NUMBER OF POPULATIONS ANALYZED: 104; 4257 MALES OUT OF 7214 INDIVIDUALS ANALYZED IN GENETIC PROFILING chromosome and to determine if Y-STR haplotype profiles were shared between individuals. Y-STR amplification was carried out in a single tube multiplex reaction [14] and genotyped on an ABI Prism™ 377 DNA Sequencer (PE Applied Biosystems, Foster City, CA). Ysingle nucleotide polymorphisms (Y-SNPs) (M89, M9, M172, 92R7, M45, M20, M70, M214, M69, M124, M173, M17) [16] were profiled hierarchically to identify the lineage of the test samples. Results and Discussion Out of the 4,257 males analyzed with either PowerPlex® 16 or Identifiler™ multiplex system, 10 confirmed male samples exhibited a dropout of the 112 bp amelogenin Yallele (Table 1). To verify the cause of observed abnormalities, we tested such samples with alternate primer pairs that encompassed the region amplified with primers reported by Sullivan et al. [6] and, are also typically used in the commercial kits. Amplification of the test samples with the primer set described by Steinlechner et al. [11] Page 4 of 7 (page number not for citation purposes) BMC Medical Genetics 2006, 7:37 http://www.biomedcentral.com/1471-2350/7/37 Table 2: Y-chromosome profiles of the amelogenin-deletion individuals* S. No Sample 1 2 3 4 5 6 7 8 9 10 Kallar Vanniyar Agharia Khatri Iyenger Brahmin Kuruva Khandayat Paroja Desasth Brahmin Maratha Y-SNP DYS 19 DYS 389I DYS 389II DYS 390 NA J2 NA NA J2 NA J2 J2 J2 J2 15 15 15 15 14 15 15 15 14 15 12 13 12 13 13 13 11 13 13 11 29 29 29 30 29 30 30 26 30 28 25 24 25 25 24 25 23 25 23 25 NA: Samples not be examined for Y-SNPs * Integers in bold represent shared haplotype between Kallar and Agahria; while those in bold italics represent haploype sharing between Khatri and Kuruva resulted in complete absence of the 225 bp Y-specific product. All 10 ambiguous samples were confirmed to be from male individuals on testing with male-specific SRY locus, which yielded the characteristic 93 bp amplicon reconfirming the gender of these subjects as males. Additional analysis with four Y-chromosomal STR markers, DYS19, DYS389I, DYS389II and DYS390, yielded complete and different Y-STR haplotype profiles. Amplification of Y-STR indicates that these samples had failed the amelogenin typing either due to mutation in the primerbinding region [17] or due to deletions in the amelogenin region (11.2p) on the Y-chromosome [11]. Among the 10 samples, eight distinct Y-STR haplotype profiles were observed; one was shared between Khatri and Kuruva and, another one by Agharia and Kallar (Table 2). Further amplification and sequencing was carried out using the forward primer of Steinlechner et al. [11] and a set of newly designed reverse primers spanning the hypothetical region of mutation – #P1, which is 62 bp downstream, and #P2, a Y-specific primer that is 43 bp downstream to the Steinlechner's reverse primer binding region, in order to determine the nature and extent of mutation. The newly designed primers depicted in Fig.2, result in 287 bp and 268 bp amplicon for #P1 and #P2 respectively, for the Y-chromosome, when used along with the forward primer of Steinlechner et al., #P1 results in a 281 bp product for the X-chromosome. However, the newly designed and validated primer sets also failed to amplify the Y-homologue in test samples suggesting deletion of a significant portion of the amelogenin region in male samples. The deletion in amelogenin gene has recently been mapped to span around 2.5 Mb [18]. The overall rate of failure among the Indian population was found to be 0.23%. Table 1 shows the frequency of failure among different endogamous groups. Failures were exhibited by both caste groups (Khatri, Desasth Brahmin, Maratha, Khandayat, Tanjore Kallar, Vanniyar and Iyenger Brahmin) and tribal populations (Agharia, Paroja, and Kuruva) while Mongoloid and Negrito populations were not found to harbor the deletions (Fig. 1). Interestingly, we observed that these deletions were present predominantly in individuals belonging to the J2 Y-chromosomal lineage. J2 is found in approximately 5.1% of the Indian population, while majority of the Indian males harbor H (25%), R1a1 (19%) and R2 (16%) haplogroups in their Y-chromosomes [19]. Probably originating in the Middle East [20], the J2 lineage has been found distributed across southeastern Europe and Asia with frequencies of 6.5% in Central Asians [16], 23.8% in Sephardic Jews, 20% in Lebanese, 17.8% in Konyan Turks, 16.3% among Italians of Apulia, 13.6% in French Basque [20], 10.2% in Moroccan Arabs [21]. The above observations and positive amplification of the SRY gene and the appearance of discrete Y-STR haplotypes, suggests that the mutations probably arose independently on a J2 Y chromosome lineage background. Of the endogamous populations screened in this study, ~10 % exhibited failures in the amelogenin gender test. Since the extent of deletion is large to avoid amplification dropout of the Y-homologue with currently available commercial primer sets, we suggest it would be prudent to include an additional gender test such as SRY and/or YSTR testing for accurate gender identification of biological specimens. Conclusion Earlier studies have reported high failure rates in amelogenin-based gender testing of individuals from the Indian sub-continent. In this study, we have analyzed 4,257 male samples and report a failure rate of 0.23%. Due to ease of typing, this test has gained wide acceptance and has been integrated into routine automated genetic profiling procedures. However, the fallibility of the amelogenin test Page 5 of 7 (page number not for citation purposes) BMC Medical Genetics 2006, 7:37 http://www.biomedcentral.com/1471-2350/7/37 Steinlechner Forward Primer Sullivan Forward Primer Sullivan Reverse Primer Steinlechner Reverse Primer Y-specific Primer Universal Primer Figure Y-chromosome tion of annealing 2 nucleotide regions of sequence the primerofsets the used human in amelogenin this study gene (GenBank Accession Number M55419) showing the locaY-chromosome nucleotide sequence of the human amelogenin gene (GenBank Accession Number M55419) showing the location of annealing regions of the primer sets used in this study. raises concern over its continued use especially in medical and forensic sciences. Although our study indicates that individuals belonging to the J2 lineage are more prone to deletion in Y-derived amelogenin gene, further corroborating studies are desired. The amelogenin-based gender test thus needs to be applied with caution, with supplementation with other Y-chromosome specific analyses for reliable gender identification. Competing interests The author(s) declare that they have no competing interests. Authors' contributions VKK conceptualized the study and contributed significantly in data interpretation and manuscript preparation. SS and TS contributed equally towards designing and carrying out of experiments, data analyses and in manuscript preparation. RT provided critical and valuable information for data processing. All authors read and approved the final manuscript. Acknowledgements This study was supported by a research grant under the X Five Year Plan to CFSL, Kolkata. The authors are thankful to Rajkumar R, Gaikwad S, Sarkar N, Tandon M, Guha S, Maity B, Ashma R, Banerjee J, Singh A and G. Hima Bindu, research scholars of the National DNA Analysis Centre, Kolkata and S. Krittika, JRF (Indian Statistical Institute, Kolkata) for providing their data for use in this study. We also thank the two reviewers for their valuable suggestions. SS and ST were assisted with MHA and CSIR fellowships, respectively. Page 6 of 7 (page number not for citation purposes) BMC Medical Genetics 2006, 7:37 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Mannucci A, Sullivan KM, Ivanov PL, Gill P: Forensic application of a rapid and quantitative DNA sex test by amplification of the X-Y homologous gene amelogenin. Int J Legal Med 1994, 106:190-193. Faerman M, Filon D, Kahila G, Greenblatt CL, Smith P, Oppenheim A: Sex identification of archeological human remains based on amplification of the X and Y amelogenin alleles. Gene 1995, 167:327-332. Findlay I, Urquhart A, Quirke P, Sullivan K, Rutherford AJ, Lilford RJ: Hum Reprod 1995, 10:1005-1013. Caenazzo L, Ponzano E, Greggio NA, Cortivo P: Prenatal sexing and sex determination in infants with ambiguous genitalia by polymerase chain reaction. Genet Test 1997, 1:289-291. Nakahori Y, Takenaka O, Nakagome Y: A human X-Y homologous region encodes "amelogenin". Genomics 1991, 9:264-269. Sullivan KM, Mannucci A, Kimpton CP, Gill P: A rapid and quantitative DNA sex test: fluorescence- based PCR analysis of XY homologous gene amelogenin. Biotechniques 1993, 15:636-641. Santos FR, Pandya A, Tyler-Smith C: Reliability of DNA-based sex tests. Nat Genet 1998, 18:103. Roffey PE, Eckhoff CI, Kuhl JL: A rare mutation in the amelogenin gene and its potential investigative ramifications. J Forensic Sci 2000, 45:1016-1019. Thangaraj K, Reddy AG, Singh L: Is the amelogenin gene reliable for gender identification in forensic casework and prenatal diagnosis? Int J Legal Med 2002, 116:121-123. Chang YM, Burgoyne LA, Both K: Higher failures of amelogenin sex test in an Indian population group. J Forensic Sci 2003, 48:1309-13. Steinlechner M, Berger B, Niederstätter H, Parson W: Rare failures in the amelogenin sex test. Int J Legal Med 2002, 116:117-120. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning. In A Laboratory manual 2nd edition. Cold Spring Harbor Laboratory Press, New York; 1989. Sinclair AH, Berta P, Palmer MS, Hawkins JR, Griffiths BL, Smith MJ, Foster JW, Frischauf AM, Lovell-Badge R, Goodfellow PN: A gene from the human sex determining region encodes a protein with homology to a conserved DNA binding motif. Nature 1990, 346:240-244. Kayser M, Caglià A, Corach D, Fretwell N, Gehrig C, Graziosi G, Heidorn F, Herrmann S, Herzog B, Hidding M, Honda K, Jobling M, Krawczak M, Leim K, Meuser S, Meyer E, Oesterreich W, Pandya A, Parson W, Penacino G, Perez-Lezaun A, Piccinini A, Prinz M, Schmitt C, Schneider PM, Szibor R, Teifel-Greding J, Weichhold G, de Knijff P, Roewer L: Evaluation of Y-Chromosomal STRs: a multicenter study. Int J Legal Med 1997, 110:125-133. Redd AJ, Clifford SL, Stoneking M: Multiplex DNA typing of short-tandem-repeat loci on the Y chromosome. Biol Chem 1997, 378:923-927. Underhill PA, Shen P, Lin AA, Jin L, Passarino G, Yang WH, Kauffman E, Bonne-Tamir B, Bertranpetit J, Francalacci P, Ibrahim M, Jenkins T, Kidd JR, Mehdi SQ, Seielstad MT, Wells RS, Piazza A, Davis RW, Feldman MW, Cavalli-Sforza LL, Oefner PJ: Y-Chromosome sequence variation and the history of human population. Nat Genet 2000, 26:358-361. Boutrand L, Egyed B, Füredi S, Mommers N, Mertens G, Vandenberghe A: Variations in primer sequences are origin of allele drop-out at loci D13S317 and CD4. Int J Legal Med 2001, 114:295-297. Lattanzi W, Di Giacomo MC, Lenato GM, Chimienti G, Voglino G, Resta N, Pepe G, Guanti G: A large interstitial deletion encompassing the amelogenin gene on the short arm of the Y chromosome. Hum Genet 2005, 116:395-401. Sahoo S, Singh A, Himabindu G, Banerjee J, Sitalaximi T, Gaikwad S, Trivedi R, Endicott P, Kivisild T, Metspalu M, Villems R, Kashyap VK: A prehistory of Indian Y Chromosomes: Evaluating demic diffusion scenarios. Proc Natl Acad Sci, USA 2006, 103:843-848. Semino O, Magri C, Benuzzi G, Lin AA, Al-Zahery N, Battaglia V, Maccioni L, Triantaphyllidis C, Shen P, Oefner PJ, Zhivotovsky LA, King R, Torroni A, Cavalli-Sforza LL, Underhill PA, Santachiara-Benerecetti AS: Origin, diffusion, and differentiation of Y-chromosome haplogroups E and J: inferences on the neolithization of Europe and later migratory events in the Mediterranean area. Am J Hum Genet 2004, 74:1023-1034. http://www.biomedcentral.com/1471-2350/7/37 21. Cruciani F, Santolamazza P, Shen P, Macaulay V, Moral P, Olckers A, Modiano D, Holmes S, Destro-Bisol G, Coia V, Wallace DC, Oefner PJ, Torroni A, Cavalli-Sforza LL, Scozzari R, Underhill PA: A back migration from Asia to sub-Saharan Africa is supported by high-resolution analysis of human Y-chromosome haplotypes. Am J Hum Genet 2002, 70:1197-1214. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2350/7/37/prepub Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." 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